Browsing by Subject "rRNA depletion"

Animal coloration is as striking as it is diverse; however, the transcriptional basis of coloration is not deeply understood. Cichlid fishes are a tractable system for studying coloration as they exhibit a wide range of phenotypic diversity while remaining genetically similar. This facilitates the study of genotype-phenotype correlations and the identification of causative genes. RNA sequencing is a powerful approach to investigate the genes which characterize chromatophores. However, RNA-seq results can be plagued by the high abundance of rRNA in cells. This thesis aims to investigate differential gene expression between differently pigmented regions as well as explore the effects of tissue treatments and rRNA depletion on gene expression. Gene sets acquired with polyA selection, riboPOOL probes optimized for zebrafish, and zebrafish probes complemented with newly designed riboPOOL cichlid probes were compared to assess the functionality of these different rRNA depletion strategies. The use of zebrafish probes complemented with newly designed cichlid probes captured the greatest diversity of genes, many transcripts of which were missing from the other gene sets. Furthermore, as experiments such as scRNA-seq rely on a dissociation step, the effect of dissociation on gene expression was examined and found to promote the expression of stress response genes. The results of this upstream optimization were applied in the analysis of differential gene expression between the vertical stripes of the cichlid Pseudotropheus demasoni to better understand the molecular basis of vertical striping in fish. The dark stripes exhibited upregulation of melanic marker genes and the light, iridescent stripes showed an increase in iridophore marker gene expression. These findings were corroborated with cell count data from FACS to link transcriptional profiles and cell type quantifications. Overall, the study provides insight into the transcriptional basis of coloration in cichlid fishes and underscores the importance of optimizing methods drawing meaningful conclusions.